The skin acts as a pathogen barrier, and the skin immune system is well-equipped for the generation of effective anti-infection immune responses. The induction of immune responses by applying a DNA vaccine topically onto the skin is an attractive immunization approach. Unfortunately, the immune responses induced using this approach is generally weak, evidently due to the difficulty for the macromolecular plasmid DNA to penetrate through the skin stratum corneum to reach the viable skin cells to allow the antigen gene(s) encoded by the plasmid to be expressed. Data from several recent studies pointed out that the hair follicles are likely to be the portal of entrance for topically applied DNA. When plasmid DNA was applied onto the skin, the expression of the gene(s) encoded by the plasmid was largely confined in the hair follicles and only occasionally in other cells in the epidermis. Moreover, the finding that the presence of normal hair follicles in the skin was required for a topical DNA vaccine to induce specific immune responses further suggested the hair follicles as the portal of entrance for topically applied plasmid DNA. Normal hair follicles cycle between growth (anagen) and resting stages. It was reported that hair follicles in the growth stage are open for penetration by foreign objects on the skin surface, whereas those in the resting stage are closed. In fact, the expression of a reporter gene applied topically onto the skin was significantly enhanced when the hair follicles in the application area were induced into anagen onset (early growth) stage. Based on all these findings, we hypothesized that the immune responses induced by a topical DNA vaccine would be enhanced when the hair follicles in the application area were induced into anagen onset stage. Using a plasmid that encodes the protective antigen (PA) protein of Bacillus anthracis as a model DNA vaccine, we have generated some promising data that were strongly supportive of this hypothesis, indicating that topical immunization by applying a DNA vaccine onto a skin area where the hair follicles are in, or induced into, anagen onset stage represents a viable immunization modality. However, before validating this approach in humans, we propose to further evaluate it by carrying out the following four specific aims: (i) to elucidate the mechanisms responsible for the enhancement of the immune response induced by a topical DNA vaccine when the hair follicles in the application area are induced into anagen (onset) stage (aim # 1), (ii) to identify factors that affect the immune responses induced by a topical DNA vaccine when the hair follicles in the application area are in, or induced into, anagen stage (aim # 2), (iii) to evaluate the extent to which the immune responses induced by a DNA vaccine using this approach will protect the animals against a lethal pathogen challenge (aim # 3), and to elucidate the mechanisms of immune induction by a DNA vaccine applied topically onto a skin area where the hair follicles are in anagen stage (aim # 4). We have assembled a team of researchers with extensive experience in vaccine development, transdermal drug delivery, skin biology, and histopathology to safeguard the successful completion of the proposed studies. The outcomes will lay a sound scientific foundation for us to improve the efficacy of topical DNA vaccines in future trials. PUBLIC HEALTH RELEVANCE: The successful completion of the proposed studies will lay a sound foundation for the development and validation of this novel topical DNA immunization approach in future (clinical) trials by modifying the hair follicle cycle. Such an efficacious non-invasive topical DNA immunization modality is expected to be amenable for mass immunization to prevent infectious diseases in humans and animals.